Cell death by apoptosis is largely responsible for control of tissue homeostastis, differentiative and immune processes. Alterations in the apoptosis program are implied in acute and chronic tissue damages (heart, kidney, brain or other tissue ischaemia, chronic degenerative disorders such as Parkinson's disease, amyotrophic lateral sclerosis and others, etc.), characterized by excessive apoptosis, and neoplastic, autoimmune and other diseases involving insufficient apoptosis. Furthermore, since antineoplastic compounds mainly act by inducing apoptosis in cancer cells, molecules involved in the apoptotic response determine neoplastic cell sensitivity or resistance to therapy. Biochemical components and/or regulators of the apoptotic pathways can be targets for modulating therapies, some of which have shown efficacy in preclinical models and are now in human clinical trials. Furthermore, apoptosis-involved molecules can represent diagnostic tools in a range of diseases and reagents for laboratory work (1).
BAG3 is member of the BAG protein family, involved in co-chaperone activity for intracellular protein folding (2). Although BAG3 displays homology with the other members of the BAG family in some portions, like the BAG domain, other parts of its nucleotide and protein sequences are unique (2-4). These BAG3-specific, unique portions have been utilised by us for the invention here described.
In the following there are reported the BAG3 nucleotide and peptide sequences; the underlined parts correspond to parts which are considered particularly relevant for the present invention.
BAG3 nucleotide sequence (SEQ ID NO: 1):
reference: NCBI PubMed. XM 055575
Homo sapiens BCL2-associated athanogene 3 (BAG3), mRNA
gi|16156810|ref|XM—055575.1|[16156810]
1gcggagctcc gcatccaacc ccgggccgcg gccaactttt ttggactgga ccagaagttt 61ctagccggcc agttgctacc tccctttatc tcctccttcc cctctggcag cgaggaggct 121atttccagac acttccaccc ctctctggcc acgtcacccc cgcctttaat tcataaaggt 181gcccggcgcc ggcttcccgg acacgtcggc ggcggagagg ggcccacggc ggcggcccgg 241ccagagactc ggcgcccgga gccagcgccc cgcacccgcg ccccagcggg cagaccccaa 301cccagcatga gcgccgccac ccactcgccc atgatgcagg tggcgtccgg caacggtgac 361cgcgaccctt tgccccccgg atgggagatc aagatcgacc cgcagaccgg ctggcccttc 421ttcgtggacc acaacagccg caccactacg tggaacgacc cgcgcgtgcc ctctgagggc 481cccaaggaga ctccatcctc tgccaatggc ccttcccggg agggctctag gctgccgcct 541gctagggaag gccaccctgt gtacccccag ctccgaccag gctacattcc cattcctgtg 601ctccatgaag gcgctgagaa ccggcaggtg caccctttcc atgtctatcc ccagcctggg 661atgcagcgat tccgaactga ggcggcagca gcggctcctc agaggtccca gtcacctctg 721cggggcatgc cagaaaccac tcagccagat aaacagtgtg gacaggtggc agcggcggcg 781gcagcccagc ccccagcctc ccacggacct gagcggtccc agtctccagc tgcctctgac 841tgctcatcct catcctcctc ggccagcctg ccttcctccg gcaggagcag cctgggcagt 901caccagctcc cgcgggggta catctccatt ccggtgatac acgagcagaa cgttacccgg 961ccagcagccc agccctcctt ccaccaagcc cagaagacgc actacccagc gcagcagggg 1021gagtaccaga cccaccagcc tgtgtaccac aagatccagg gggatgactg ggagccccgg 1081cccctgcggg cggcatcccc gttcaggtca tctgtccagg gtgcatcgag ccgggagggc 1141tcaccagcca ggagcagcac gccactccac tccccctcgc ccatccgtgt gcacaccgtg 1200gtcgacaggc ctcagcagcc catgacccat cgagaaactg cacctgtttc ccagcctgaa 1261aacaaaccag aaagtaagcc aggcccagtt ggaccagaac tccctcctgg acacatccca 1321attcaagtga tccgcaaaga ggtggattct aaacctgttt cccagaagcc cccacctccc 1381tctgagaagg tagaggtgaa agttccccct gctccagttc cttgtcctcc tcccagccct 1441ggcccttctg ctgtcccctc ttcccccaag agtgtggcta cagaagagag ggcagccccc 1501agcactgccc ctgcagaagc tacacctcca aaaccaggag aagccgaggc tcccccaaaa 1561catccaggag tgctgaaagt ggaagccatc ctggagaagg tgcaggggct ggagcaggct 1621gtagacaact ttgaaggcaa gaagactgac aaaaagtacc tgatgatcga agagtatttg 1681accaaagagc tgctggccct ggattcagtg gaccccgagg gacgagccga tgtgcgtcag 1741gccaggagag acggtgtcag gaaggttcag accatcttgg aaaaacttga acagaaagcc 1801attgatgtcc caggtcaagt ccaggtctat gaactccagc ccagcaacct tgaagcagat 1861cagccactgc aggcaatcat ggagatgggt gccctggcag cagacaaggg caagaaaaat 1921gctggaaatg cagaagatcc ccacacagaa acccagcagc cagaagccac agcagcagcg 1981acttcaaacc ccagcagcat gacagacacc cctggtaacc cagcagcacc gtagcctctg 2041ccctgtaaaa atcagactcg gaaccgatgt gtgctttagg gaattttaag ttgcatgcat 2101ttcagagact ttaagtcagt tggtttttat tagctgcttg gtatgcagta acttgggtgg 2161aggcaaaaca ctaataaaag ggctaaaaag gaaaatgatg cttttcttct atattcttac 2221tctgtacaaa taaagaagtt gcttgttgtt tcagaagttt aaccccgttg cttgttctgc 2281agccctgtct acttgggcac ccccaccacc tgttagctgt ggttgtgcac tgtcttttgt 2341agctctggac tggaggggta gatggggagt caattaccca tcacataaat atgaaacatt 2401tatcagaaat gttgccattt taatgagatg attttcttca tctcataatt aaaatacctg 2461actttagaga gagtaaaatg tgccaggagc cataggaata tctgtatgtt ggatgacttt 2521aatgctacat ttt
BAG3 aminoacidic sequence (SEQ ID NO: 2):
reference: NCBI PubMed. XM 055575
Homo sapiens BCL2-associated athanogene 3 (BAG3), mRNA
gi|16156810|ref|XM—055575.1|[16156810]
MSAATHSPMMQVASGNGDRDPLPPGWEIKIDPQTGWPFFVDHNSRTTTWNDP RVPSEGPKETPSSANGPSREGSRLPPAREGHPVYPQLRPGYIPIPVLHEGAENR QVHPFHVYPQPGMQRFRTEAAAAAPQRSQSPLRGMPETTQPDKQCGQVAAAA AAQPPASHGPERSQSPAASDCSSSSSSASLPSSGRSSLGSHQLPRGYISIPVIHE QNVTR PAAQPSFHQAQKTHYPAQQGEYQTHQPVYHKIQGDDWEPRPLRAASPFRSSVQ GASSREGSPARSSTPLHSPSPIRVHTVVDRPQQPMTHRETAPVSQPENKPESKP GPVGPELPPGHIPIQVIRKEVDSKPVSQKPPPPSEKVEVKVPPAPVPCPPPSPGPS AVPSSPKSVATEERAAPSTAPAEATPPKPGEAEAPPKHPGVLKVEAILEKVQGLEQ AVDNFEGKKTDKKYLMIEEYLTKELLALDSVDPEGRADVRQARRDGVRKVQTILEK LEQKAIDVPGQVQVYELQPSNLEADQPLQAIMEMGAVAADKGKKNAGNAEDPHT ETQQPEATAAATSNPSSMT DTPGNPAAP
BAG3 protein is known to be expressed in some cell lines, such as HeLa and A2058, and, as far as normal primary human cells are concerned, in skeletal muscle, heart, ovary and other types of normal cells (2-5). BAG3 expression has also been detected in human pancreas tumour cells (6).
BAG3 expression had not been reported in other types of primary normal or neoplastic cells before the results here reported for the first time.
Some findings describe that transfection of cells of the human cell line HeLa (5) or of the murine cell line 32D (7) with BAG3 hyperexpressing constructs can modestly increase cell apoptosis induced by Bax microinjection or via Fas (5), or by IL-3 deprivation (7), respectively.
Generically antibodies for BAG3 have been described in WO00/14106 and WO95/25125, however there has not been characterized any immunogenic site specific for them. Ref.s 4-6 describe polyclonal antibodies specific for the carbossi-terminal region of BAG3 protein starting from amino acid 306 specifically. Liao describes a rabbit polyclonal anti-BAG3 antibody against the 196 amino acids of the C-terminal portion of BAG3. Lee describes a polyclonal antibody against the amino acid region encompassing the portion 306-575. Dong describes a polyclonal antibody against the two amino acid regions 2 and 8.
Patent abstract of Japan publication 10327872 describes uses of BAG3 for diagnosis, prophylaxis and therapy of pathologies relating to apoptosis, however there has not been characterized any immunogenic site or any specific antibody, moreover test, in particular in humans, are absent.
Before results here reported for the first time, BAG3 expression had not been proved to influence apoptosis in human primary cells, either normal, neoplastic or affected by other types of pathologies. Furthermore, BAG3 downmodulation by reagents, such as oligonucleotides, that can be used in primary cells, and its effects on cell apoptosis had never been reported.